The ability to regulate gene expression in vivo in transgenic animals, including humans, is of vital importance both to the investigation of gene function and to the control and utility of therapeutic gene expression in gene therapy in animals or humans.
A variety of approaches have been attempted to develop a reliable system for controlling gene expression in vivo. The first attempts were based on the use of promoters that could be induced by endogenous transcription factors in response to a controllable stimulus such as heat shock (Wurm F. M., Gwinn K. A. and Kingston R. E., “Inducible overproduction of the mouse c-myc protein in mammalian cells” Proc. Natl. Acad. Sci. USA 83: 5414-5418 (1986)) or heavy metal ions (Mayo K. E., Warren R. and Palmiter R. D., “The mouse metallothionein-1 gene is transcriptionally regulated by cadmium following transfection into human or mouse cells” Cell 29: 99-108 (1982)). However, it was found that induction ratios were low and the induction agent itself often activated a large number of unwanted endogenous genes.
To overcome these problems, attempts were made to develop inducible systems which use chimeric transcription factors that combine elements from mammalian, bacterial, yeast and viral transcription factors (for review, see Gossen M., Bonin A. L. and Bujard H., “Control of gene activity in higher eukaryotic cells by prokaryotic regulatory elements” Trends in Biochem. Sci. 18: 471-475 (1993)).
One example of the use of these systems is the use of the lac repressor, which can then be induced by isopropyl D-thiogalactopyranoside (IPTG) (Baim S. B., Labow M. A., Levine A. J. and Shenk T., “A chimeric mammalian transactivator based on the lac repressor that is regulated by temperature and isopropyl D-thiogalactopyranoside” Proc. Natl. Acad. Sci. U.S.A. 88: 5072-5076 (1991)). This system is seriously limited by the toxicity of IPTG in animals.
In an alternative system, the DNA binding domain of the tetracycline (tet) repressor from E. coli is combined with the activating domain of the herpes simplex virus protein VP16 (Gossen M. and Bujard H., “Tight control of gene expression in mammalian cells by tetracycline-responsive promoters” Proc. Natl. Acad. Sci. U.S.A. 89: 5547-5551 (1992)). The gene of interest is placed downstream of the multiple tet operator sequences. In the absence of tetracycline, the tet/VP 16 activator will bind the operator sequence and activate the downstream gene. In the presence of tetracycline, the gene of interest will not be transcribed because the binding of the tet/VP16 activator will be inhibited. This system has been shown to have the ability to control reporter gene expression in vivo in transgenic mice (Furth, P. A., St. Onge L., Boger H., Gruss P., Gossen M., Kistner A., Bujard H. and Hennighausen L., “Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter” Proc. Natl. Acad. Sci. U.S.A. 91: 9302-9306 (1994)). However, this system and its variants suffer from the serious disadvantage that tetracycline is used as the repressor and must always be present to keep the downstream gene of interest silent. In addition, the bacterial protein components may be immunogenic in humans.
The disadvantages of the systems described above have provided incentives for an entirely different approach to the control of therapeutic gene expression in, for example, gene therapy. This approach creates an inducible gene control system by fusing the hormone binding domain (HBD) or ligand binding domain (LBD) of a steroid hormone receptor with certain proteins (Wang Y., O'Malley B. W., Jr., Tsai S. Y. and O'Malley B. W., “A regulatory system for use in gene transfer” Proc. Natl. Acad. Sci. 91: 8180-8184 (1994)). In such a fusion product, a number of proteins will be inactive in the absence of hormone but resume normal activity in the presence of the hormone or hormone variant which binds at this domain.
The proteins used in such a system may have a wide variety of thus controllable activities. For example, these proteins may be regulator proteins specific for the control of the transcription of particular transgene. Many control systems of this type have been constructed using the HBD of the estrogen receptor (ER) (Hollenberg S. M., Cheng P. F. and Weintraub H., “Use of conditional MyoD transcription factor studies of MyoD transactivation and muscle determination” Proc. Natl. Acad. Sci. U.S.A. 90: 8028-8032 (1993); Braselmann S., Graninger P. and Busslinger M., “A selective transcriptional induction system for mammalian cells based on GAL4-estrogen receptor fusion proteins” Proc. Natl. Acad. Sci. 90: 1657-1661 (1993); Roemer K. and Friedmann T., “Modulation of cell proliferation and gene expression by a p53-estrogen receptor hybrid protein” Proc. Natl. Acad. Sci. USA 90: 9252-9256 (1993); Superti-Furga G., Bergers G., Picard D. and Busslinger M., “Hormone-dependent transcriptional regulation and cellular transformation by Fos-steroid receptor fusion proteins” Proc. Natl. Acad. Sci. U.S.A. 88: 5114-5118 (1991)). The ligand of the ER, 17β-estradiol is readily available, relatively cheap and many cell types lack an endogenous estrogen receptor. However, these systems employing the HBD of the wildtype estrogen receptor are potentially disadvantageous in that use of the hormone to control the inducible system will also activate endogenous steroid hormone receptors and thereby alter the activity of endogenous genes. Furthermore, the systems will be influenced by levels of endogenous β-estradiol.
One approach to regulating transgene expression and avoiding the activation of endogenous and unwanted genes has been the modification and use of chimeric nuclear hormone receptors, such as a steroid hormone receptor. Steroid hormone receptors are responsible for the regulation of complex cellular events, including transcription. The ovarian hormones, estrogen and progesterone, are responsible, in part, for the regulation of the complex cellular events associated with differentiation, growth and functioning of female reproductive tissues. These hormones also play important roles in development and progression of malignancies of the reproductive endocrine system.
The biological activity of steroid hormones is mediated directly by a hormone and tissue-specific intracellular receptor. The physiologically inactive form of the steroid receptor may exist as an oligomeric complex with proteins, such as heat-shock protein (hsp) 90, hsp70 and hsp56. Upon binding its specific ligand, the receptor changes conformation and dissociates from the inhibitory heteroligomeric complex. Subsequent dimerization allows the receptor to bind to specific DNA sites in the regulatory region of target gene promoters. Following binding of the receptor to DNA, the hormone is responsible for mediating a second function that allows the receptor to interact specifically with the transcription apparatus. Displacement of additional inhibitory proteins and DNA-dependent phosphorylation may constitute the final steps in this activation pathway.
Cloning of several members of the steroid receptor superfamily has facilitated the reconstitution of hormone-dependent transcription in heterologous cell systems. Subsequently, in vivo and in vitro studies with mutant and chimeric receptors have demonstrated that steroid hormone receptors are modular proteins organized into structurally and functionally defined domains. A well-defined 66-68 amino acid DNA binding domain (DBD) has been identified and studied in detail, using both genetic and biochemical approaches. The ligand (hormone) binding domain (LBD), located in the carboxyl-terminal half of the receptor, consists of about 300 amino acids. Thus, these nuclear receptors, such as the estrogen receptor (ER), are ligand-activated transcription factors that include a DNA binding domain (DBD) and a hormone binding domain (HBD) also known as a ligand binding domain (LBD). The LBD also contains sequences responsible for receptor dimerization, hsp interactions and one of the two transactivation sequences of the receptor. See, for example, Nilsson et al., “Mechanisms of Estrogen Action” Physiol. Rev. 81(4): 1535-1565 (2001), incorporated herein by reference.
In order to make a system utilizing these elements as adaptable and useful as possible, these chimeric regulator proteins are preferably altered in their DNA binding specificity, to make them specific for control of the desired transgene (see WO 01/30843A1, which is incorporated by reference herein for all purposes) and they are preferably altered in their ligand binding specificity so they will respond to a ligand that will not cause altered activity of other endogenous genes.
For example, a preferred pharmacological profile for the LBD of the chimeric regulator would include the following features:    1) Physiologic levels of endogenous hormones normally found in man do not activate the receptor;    2) The receptor could be activated by a custom designed, synthetic compound at levels (doses) that can be achieved in vivo without toxicity; and    3) At the required dose for transgene regulation, the synthetic compound is inactive, i.e., is neither an agonist nor antagonist, on the naturally occurring endogenous hormone receptors.
Gene replacement therapy requires the ability to control the level of expression of transfected genes from outside the body. Such a “molecular switch” preferably includes the properties of: specificity, selectivity, precision, safety and rapid clearance. The steroid receptor family of gene regulatory proteins is an ideal set of such molecules. These proteins are ligand activated transcription factors whose ligands can range from steroids to retinoids, fatty acids, vitamins, thyroid hormones and other specifically engineered small molecules. These compounds bind to receptors and either up-regulate or down-regulate. The compounds are cleared from the body by existing mechanisms and the compounds are non-toxic.
The efficacy of a ligand is a consequence of its interaction with the receptor. This interaction can involve contacts causing the receptor to become active (agonist) or for the receptor to be inactive (antagonist). The affinity of antagonist activated receptors for DNA is similar to that of agonist-bound receptor. Nevertheless, in the presence of the antagonist, the receptor cannot activate transcription efficiently. Thus, both up and down regulation are possible by this pathway.
Modified steroid hormone receptors have been developed for use for regulated expression of transgenes (see, e.g., U.S. Pat. Nos. 5,874,534 and No. 5,935,934 and PCT publication No. WO 98/18925, which claims priority to U.S. Provisional Application No. 60/029,964) by modifying the ligand specificity of the LBD. In addition, the DNA binding domain of the receptor has been replaced with a non-mammalian DNA binding domain selected from yeast GAL4 DBD, a viral DBD and an insect DBD binding domain to provide for regulated expression of a co-administered gene containing a region recognized by the non-mammalian DBD. These constructs, however, have several potential drawbacks and generally lack flexibility. The non-mammalian DBD is potentially immunogenic and the array of sequences recognized by these DBD's is limited, accordingly limiting gene targets. Therefore, there remains a need for versatile and effective gene regulators.